As part of normal environment testing there is an on-going need for methods of rapidly detecting and quantifying the presence of target molecules. For example, small molecules such as endocrine disrupting compounds and hormones are often found as contaminants in the environment. Such contaminants can be found in waterways, soils, biological samples, including both plant and animals, as environmental pollutants from residential, agricultural, commercial and/or industrial applications. It is known, in some cases, that these small molecular weight compounds, such as those indicated below, together with their metabolites and/or synthetically modified variants pose a threat to the health of human and wildlife populations by mimicking the activity of endogenous hormones such as oestrogen. These molecules may act by blocking, mimicking, stimulating or inhibiting the production and function of natural hormones. The organic residues that mimic these endogenous steroidal hormones, and metabolites are lipid soluble, thus have the ability to bio-accumulate in living systems of mammals and marine species. Evidence of this has been identified in human blood plasma, breast milk, foetal tissues and biological fluids [Allmyr et al., Anal. Chem., 78: 6542-6546, 2006; Hileman, Chemical and Engineering News, 85: 31-33, 2007; Van-Pelt et al. Endocrinology, 140: 478-483, 2001; Skakkebaek et al., Human Reproduction 16: 972-978, 2001; Vandenberg et al., Endocrine Reviews, 33(3): 2012] Therefore, there is a need for new methods for easy detection of these small molecules.

More conventional methodologies and techniques that are often used for the detection of small compounds include High Performance Liquid Chromatography (HPLC) or Gas Chromatography coupled with Mass Spectrometry (GCMS). These techniques are very useful for this purpose but the analyses can be complicated to perform and can take a long period of time to complete. Therefore, these techniques cannot be performed on site, they require specialised equipment and trained operators, and do not provide for a rapid assessment.
There is therefore a need for a convenient, quick and simple method for the detection and quantification of small molecules, especially in the area of environmental and contaminant testing.
Aptamers are single-stranded nucleic acids (ssRNA, ssDNA), which unlike traditional nucleic acids, possess unique binding characteristics to specific targets with high affinity and specificity analogous to antibodies [Tuerk, C. Gold, L., Science, 1990, 249(4968), 505-510; Ellington, A. D., Szostak, J. W., Nature, 1990, 346(6287), 818-822.] Aptamers are isolated in vitro from combinatorial oligonucleotide libraries, typically containing 1012 to 1015 oligonucleotides, and are chemically synthesised by a process known as SELEX. The oligonucleotides are subjected to a selection process for their ability to bind a specified target and over a number of selection rounds (typically 8-16 rounds); the most specific nucleic acid sequences are isolated. Depending on the techniques used in SELEX, the process might take from days to months [Cho, E. J., Lee, J. W., Ellington, A. D., Ann. Rev. Anal. Chem., 2009, 2(1), 241-264; Ellington, A. D., Ann. Rev. Anal. Chem., 2009, 2(1), 241-264.] Aptamers have been generated for a wide range of targets, ranging from ions to entire cells. The use of an in vitro process enables the generation and selection of aptamers that can bind toxic targets, which are not possible by immunologically initiated recognition elements, such as antibodies. The small size of aptamers (generally <3 nm in a coiled conformation) also makes them more readily applicable to surface-based aqueous sensing purposes in comparison to antibodies (approximately >10 nm in size) [Song, S., et al., Trends in Analytical Chemistry, 2008, 27(2), 108-117].
Baker et al., [Olowu, R. A.; Arotiba, O.; Maliu, S. N.; Waryo, T. T.; Baker, P.; Iwouoha, E., Sensors, 2010, 10, 9872] have previously shown that electrochemical DNA aptasensors developed from poly(3,4-ethylenedioxythiophene) (PEDOT) doped with gold nanoparticles (AuNP) have high affinity for the detection of 17β-estradiol. The PEDOT-AuNP are synthesised for the immobilisation of 17β-estradiol. This PEDOT-AuNP is able to reliably detect 17β-estradiol in the range of 0.1 nM-100 nM, with a detection limit of 0.02 nM.
In addition, Baker et al. [Olowu, R. A., Ndangili, P. M., Baleg, A. A, Ikpo, C. O., Njomo, N., Baker, P, Iwuoha, E., Int. J. Electrochem. Sci., 2011, 6, 1686] have also prepared and shown an aptamer biosensor developed from a dendritic first generation poly(propyleneimine)-polythiophene copolymer (shown below)-functionalised gold electrode via biotin-avidin interaction in the determination of endocrine disrupting compounds, especially 17β-estradiol.

The sensor platform and aptasensor were investigated using techniques such as scanning electron microscopy, Fourier transform infrared spectroscopy, electrochemical impedance spectroscopy, cyclic voltammetry and square wave voltammetry. The authors report that the response in the detection of 17β-estradiol was measured using square wave voltammetry with a linear range of the sensor of 0.1 to 100 nM. In addition, this particular aptamer is specific only to 17β-estradiol.
Lee and Gu et al. [Kim, Y. S., Jung, H. S., Matsuura, T., Lee, H. Y., Kawai, T., Gu, M. B. Biosensors and Bioelectronics, 2007, 22, 2525] report the synthesis and use of ssDNA aptamer based electrochemical biosensors by immobilisation of the ssDNA aptamer on a gold electrode chip in the detection of 17β-estradiol. The detection levels of 17β-estradiol are reported to be in the range of from 1000 to 0.1 nM. However, the authors report at lower concentrations, in the range of 0.01 nM to 0.001 nM, their measurements may not be related to the binding of the aptamer to the substrate which makes this particular method unreliable.
US 2012/0088232 teaches a method for the detection of target molecules in patient samples at a point of care location using a point of care lateral flow device. The point-of-care lateral flow device specifically detects cancer markers and proteins, in particular p-glucoprotein (Pgp), by utilising aptamers that have been labelled with appropriate tags such as fluorophores. The aptamers are conjugated to solid supports, such as nanoparticles, and the presence of the target substrate molecules are quantified using techniques, including dynamic light scattering. In this case, dynamic light scattering measures increases in particle sizes associated with aptamer-substrate complex formation.
Any reference to prior art publications within this specification does not constitute an admission that such references form part of the common general knowledge in the art in any country.
It is an object of the present invention to provide a method for the rapid detection of small molecules in a sample, or to at least provide the public with a useful alternative.
The inventors of the present invention have surprisingly found that NP-aptamer-conjugates disclosed herein can be successfully applied to the detection of small molecules at low levels providing for quick confirmation of the presence of these small molecules in isolated samples.